Changes in oxygen tension modulate osteoblast gene expression and long-term differentiation

Abstract

Introduction: Disruption of blood flow with bony and soft-tissue injury results in a hypoxic gradient within the wound microenvironment. As a first step to understanding osteoblast behavior following hypoxic insult, we perform large-scale transcriptional profiling of hypoxic and anoxic osteoblasts. In addition, we investigate long-term osteoblast differentiation after brief hypoxic exposure. Methods: RNA from murine calvarial osteoblasts cultured in 21%, 2%, or 0% O2 for up to 24 hours was used for microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Osteoblasts exposed to 24 hours of hypoxia were differentiated for 28 days and compared to non-hypoxic cultures via qRT-PCR and bone-nodule staining by the Von Kossa method. Results: Osteoblast exposure to 0% or 2% O2 for 24 hours resulted in statistically significant expression changes for approximately 700 genes in each group. Of these, only 89 genes were shared by both groups. We observed differential regulation of osteogenic and angiogenic markers, transcription and growth factors, and inflammatory signaling modulators. As expected, both conditions induced anaerobic glycolysis, but only anoxia elevated heat-shock response elements. Surprisingly, osteoblasts exposed to 24 hours of anoxia demonstrated diminished bone nodule formation at 28 days. Conclusions: Gene expression changes in osteoblasts within the hypoxic post-injury microenvironment remain largely unknown. Transcriptional profiling reveals distinct patterns of expression at 0% versus 2% O2 relative to ambient oxygen. Furthermore, in vitro osteoblast differentiation is diminished even after brief anoxic insult. Together with induction of angiogenic and inflammatory genes, this underscores the involvement of additional cell types for successful bone repair.